MS-Net: Multi-Similarity based network annotation for untargeted metabolomics: Raw LC-MS data from Cannabis extract

Raw LC-MS data in POS and NEG mode acquired from Cannabis extracts to illustrate MS-Net workflow.

Acquisition method:

Chromatographic separation was carried out on a Vanquish UHPLC system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a Luna Omega Polar C18 analytical column (150 × 2.1 mm, 1.6 µm; Phenomenex, Torrance, CA, USA). The chromatographic system was coupled with a Vanquish Diode Array Detector (DAD), a Charged Aerosol Detector (CAD) and a Q-exactive Plus mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA). A dual-pump system was installed, where the first pump managed the separation at the column Level, and the second pump system generated a counter-gradient at the column outlet to maintain a constant mixture of 50% water and 50% acetonitrile. The separation was performed at a flow rate of 0.4 mL·min⁻¹ and at a column oven temperature of 40 °C, using a mobile phase composed of H₂O + 0.05% formic acid (A) and acetonitrile + 0.05% formic acid (B). A 1 µL injection of extracts at 10 mg·mL⁻¹ was introduced into the system. The applied gradient was as follows: 0 to 0.5 min, 2% B; 0.5 to 18 min, 98% B; 18 to 21 min, 98% B; 21 to 21.5 min, 2% B; and 21.4 to 24 min, 2% B.

Mass spectrometry detection was performed using a Q Exactive Plus instrument equipped with a heated electrospray ionization source (HESI-II) operating in positive and negative ionization modes with a resolution of 35,000 (MS1) and 17,500 (MS2). The collision energy was set to 10, 20, and 40 eV in stepped mode for data-dependent MS/MS acquisition. The capillary temperature was 300°C, and the mass scan range was 100–1,500 m/z for both MS1 and MS2. Data-dependent acquisition targeted the four most intense precursor ions per scan cycle.